Conceived and designed the experiments: JS CK. Performed the experiments: CK SD. Analyzed the data: JS CK SD AS. Wrote the paper: JS CK AS.
The authors have declared that no competing interests exist.
In asthma, mechanisms contributing to chronicity remain to be determined. Recent models of sensitisation with prolonged airway allergen challenges reproduce typical features of chronic asthma. However, the interplay between inflammation, structural changes and lung function is poorly understood. This study was performed to delineate functional, structural and immunological airway changes after cessation of long term challenges to elucidate factors contributing to the development of prolonged lung function changes.
Mice sensitised systemically were consecutively challenged intranasally with ovalbumin for two or eight weeks. After the end of challenges, lung function, airway inflammation, features of airway remodelling, local T-cell cytokines and systemic ovalbumin-specific antibodies were monitored. Long term challenges resulted in airway hyperresponsiveness lasting 2 weeks and reduced baseline lung function for 6 weeks after their cessation. In contrast, these changes resolved within one week after short term challenges. Prolonged transforming growth factor beta (TGF-β)1 production and marked peribronchial fibrosis were only induced by long term challenges. Importantly, fibrosis became apparent only after the onset of lung function changes and outlasted them. Further, long term challenges led to prolonged and intense airway inflammation with marked lymphocytosis, but moderate eosinophilia, sustained IL-5 production and ovalbumin-specific IgG2a antibodies, the latter suggesting a Th1 component to the immune response. In contrast, following short term challenges airway inflammation was dominated by eosinophils and associated with a strong, but transient IL-13 response.
Prolonged lung function changes after long term allergen challenges seem to develop and resolve independently of the persistent peribronchial fibrosis. They are more closely associated with intense airway inflammation, marked lymphocytosis, prolonged IL-5 and TGF-β1 production in the airways and a Th1 immune response.
Asthma now affects more than 10% of children in industrialized countries
Mouse models of allergic airway sensitisation with short term allergen challenges have elucidated mechanisms of acute inflammatory and functional responses like airway eosinophilia and transient airway hyperresponsiveness (AHR) (reviewed by Kumar
Predominance of Th2 cytokines in murine models of acute allergic airway inflammation is well established
Here, we report a sensitisation model with long term airway allergen challenges which induce typical features of chronic asthma including protracted changes in lung function, airway inflammation, mucus hyperplasia and peribronchial fibrosis. To delineate factors contributing to these chronic changes we compared immunological, functional and structural consequences after the cessation of long and short term challenges.
Female BALB/c mice, 6–8 weeks of age, from Charles River Deutschland (Sulzfeld, Germany) were used under protocols approved by Regierungspräsidium Arnsberg (NRW, Germany).
Mice were sensitised on days 1 and 7 by intraperitoneal injection of 20 µg ovalbumin (OVA) (Sigma-Aldrich, Taufkirchen, Germany) emulsified in 2.25 mg aluminium hydroxide (AlumImuject; Pierce, Rockford, Ill) in 100 µl. Mice were challenged intranasally with 40 µl of 1% OVA in PBS under light anaesthesia with xylazine (BayerVital, Leverkusen, Germany)/ketamine (CuraMED Pharma, Karlsruhe, Germany). Controls were treated intraperitoneally with PBS, emulsified in aluminium hydroxide, and intranasally with sterile PBS. Two different challenge protocols were used: once weekly airway challenges for 2 weeks (short term) or for 8 weeks (long term). 48 hours, 2, 4, and 8 weeks after the final airway challenge lung function was determined. 24 hours after lung function tests, mice were sacrificed for harvest of bronchoalveolar lavage (BAL) fluid, lungs and serum. Blood was drawn by tail vein puncture prior to cervical dislocation.
Baseline lung function and airway responsiveness to increasing concentrations of methacholine (MCh) (Sigma-Aldrich) were assessed using a single-chamber, whole-body plethysmograph (Buxco Electronics Inc, Troy, New York, USA) as described
Lungs were fixed with 4% paraformaldehyde prior to embedding in paraffin. 5 µm sections were stained with haematoxylin/eosin to assess inflammatory infiltrates, with Alcian Blue Periodic Acid -Schiff (PAS) to detect mucin in goblet cells, and with Masson's trichrome to determine the amount of peribronchial collagen (all dyes from Merck, Darmstadt, Germany). Sections were analysed using a Zeiss Axioplan 2 imaging microscope, Axiocam camera and Axiovision 4.3 software (Zeiss, Jena, Germany). Airway inflammation was quantified in the peribronchial region of 6–8 different medium-sized bronchi per slide at ×20 magnification, using a semi-quantitative scoring system with a grading scale from 0 (no inflammation) to 4 (very severe inflammation). Goblet cell hyperplasia was determined by enumerating PAS-positive cells in airway epithelium at ×40 magnification. Collagen deposition (green stain) was quantified in peribronchial areas in sections stained with Masson's trichrome using AnalySis 3.2 software (Software Imaging Systems, Münster, Germany) by determining the ratio of green area per total tissue/matrix area (green/100-non-stained).
Total leukocyte numbers in the BAL fluid were counted in a Neubauer chamber. Following cyto-centrifugation (Cytospin, Shandon Inc., Pittsburgh, PA) and staining with Haema Schnellfärbelösung, (Labor und Technik, Eberhard Lehmann, Berlin, Germany), at least 300 cells per slide were differentiated by a blinded investigator using standard haematological criteria.
Cytokine levels in BAL fluid were measured by ELISA for IL-4, IL-10 and IL-12 (OptEIA, Becton Dickinson, Heidelberg, Germany) as well as for IL-13 and activated TGF-β1 (Quantikine Sandwich ELISA, R&D Systems, Wiesbaden, Germany) according to the manufacturer's directions. In IL-5 and IFNγ ELISA the following antibodies were used: Purified rat anti-mouse IL-5 (TRFK5) for coating and biotin rat anti-mouse IL-5 (TRFK4) for detection, and rat anti-mouse IFNγ (XMG1.2) purified and biotinidated for coating and detection (all Pharmingen, Becton Dickinson, Heidelberg, Germany). The detection limits were 7 pg/ml for IL-4, 15 pg/ml for IL-5, 8pg/ml for IL-10, 80 pg/ml for IL-12p70, 5 pg/ml for IL-13, 20 pg/ml for IFNγ and 30pg/ml for activated TGF-β1.
Lungs were minced, digested with collagenase (Sigma-Aldrich) for 30 min, and washed. Mononuclear cells were isolated by Ficoll (Biochrom AG, Berlin, Germany) gradient. For intracellular cytokine staining, lung cells were stimulated for 4 hours with PMA (30 ng/ml), and ionomycin (300 ng/ml) in the presence of brefeldin A (10 µg/ml) (all from Sigma-Aldrich). Following fixation and permeabilization, cells were stained with the appropriate antibodies or isotype controls. A FACScan analyser (Becton Dickinson) was used for data acquisition and Cellquest software (Becton Dickinson) for analysis. The following antibodies (all Becton Dickinson) were used: anti-CD3 (145-2C11) FITC-conjugated; anti-IL-4 (11B11), anti-IL-5 (TRFK5), and anti-IFNγ (XMG1.2) PE-conjugated, rat IgG2a (R35-95), rat IgG2b (A95-1), rat IgG1 (R3-34), mouse IgG2b (MPC-11) and hamster IgG (A19-3) were used as isotype controls.
Serum levels of OVA-specific IgE, IgG1, and IgG2a were measured by ELISA as described. Briefly, 96-well-plates (Greiner bio-one, Frickenhausen, Germany) were coated with OVA (5 µg/ml) (Sigma-Aldrich) in carbonate buffer and washed. Then serum samples were added and plates were washed again. For IgE detection, biotinylated anti-IgE antibody (R35-72, Becton Dickinson) was used with extravidin-POD (Sigma-Aldrich) and TMB as substrate. OVA-specific IgG1 and IgG2a were detected with alkaline phosphatase-labeled anti-IgG1 (X56) or anti-IgG2a (R19-15) antibodies (both Becton Dickinson) respectively and pNPP substrate (Sigma-Aldrich). OVA-specific antibody titres were related to an internal, pooled standard and expressed as internal units (IU)/ml. The detection limit for OVA-specific IgE was 2,0 IU/ml, for OVA-specific IgG1 and –IgG2a 0,001 IU/ml.
Data were compared using GraphPad Prism 4.02 (GraphPad Software, San Diego, USA). Mann-Whitney test was used for comparison of 2 groups and Kruskal-Wallis test with Dunn post hoc test were used for comparisons of more than 2 groups. P values for significance were set at 0.05 except for flow cytometry data and Penh data where p values of <0.01 were regarded as significant. Values are expressed as mean±SEM for all measurements.
Following short term as well as long term OVA challenges in OVA sensitised mice, AHR of comparable magnitude developed. 48 hours after the last challenge maximal Penh values at 50 mg/ml MCh were 7.38±0.72 and 6.74±0.37 for long term and short term challenges respectively, compared to 3.96±0.26 in PBS challenged controls. After short term challenges AHR resolved within one week, while it persisted for 2 weeks after long term challenges (
(a) Following OVA sensitisation and short or long term OVA challenges or after sham sensitisation and long or short term PBS challenges, airway responsiveness to MCh was assessed 48 hours after final airway challenge and then weekly for 8 weeks. Mean±SEM of Penh values at 50 mg/ml MCh are shown for the first 4 weeks only from 3 independent experiments (n≥12). No differences between groups were detected after week 4. (b) Baseline Penh values were assessed during the same measurements. Mean±SEM from 5 independent experiments are illustrated (n≥15). Significant differences: * long term OVA challenges versus PBS control, +short term OVA challenges versus PBS control, levels of significance: */+ p<0,01, **/++ p<0,001.
Both short and long term airway OVA challenges induced marked airway inflammation (
Representative photomicrographs of paraffin-embedded lung sections stained with H&E (left column), Alcian-PAS (middle column) and Masson's trichrome (right column) from short term OVA challenged animals (d–l), long term OVA challenged animals (m–u) and after PBS treatment (a–c) 3 days (row 2 and 5), 4 weeks (row 3 and 6) and 8 weeks (row 4 and 7) after challenges. Magnification: 20-fold for H&E, 20-fold for Masson's trichrome, and 40-fold for Alcian-PAS stained sections.
To characterise the quality of airway inflammation, BAL cells were analysed. 72 hours after the final challenge total cell numbers in BAL fluid were raised 6–10 fold compared to control mice without significant differences between the two challenge protocols (PBS: 124,900±18,300 cells/ml, short term OVA: *1,081,100±244,900, long term OVA: *841,800±90,200, *p<0.05 versus PBS, n≥10). BAL cell numbers remained significantly elevated (3-fold over controls) until 8 weeks after the final challenge following long term challenges, whereas they returned to baseline within 2 weeks following short term challenges (data not shown). OVA challenges resulted in eosinophilia and lymphocytosis in BAL fluid, but with striking differences between protocols (
Following OVA sensitisation and short or long term OVA challenges or after sham sensitisation and long or short term PBS challenges, numbers of (a) eosinophils, and (b) lymphocytes were determined in BAL fluid from 3 days to 8 weeks after final airway challenge, n≥12 per group from 5 independent experiments. Significant differences: * OVA challenges versus PBS controls, +long term versus short term OVA challenges, */+ p<0,05, **/++ p<0,01, ***/+++ p<0,001.
OVA challenges in both protocols resulted in pronounced goblet cell hyperplasia 72 hours after final challenges (
Increased airway collagen deposition, a typical feature of airway remodelling in chronic asthma, was assessed peribronchially comparing long term to short term airway challenges. By 4 weeks after long term OVA challenges the collagen matrix around the bronchi was increased 2 to 3-fold to *8.1±4.1% (n = 9, *p<0.05) (
Th2 cytokines (IL-4, IL-5 and IL-13), which dominate many murine models of short term airway sensitisation, Th1cytokines (IFN-γ and IL-12), as well as IL-10, a cytokine associated with regulatory immune responses, were measured in BAL fluid by ELISA. Both short and long term OVA challenges induced increased levels of IL-5 and IL-13, however with marked differences between the two protocols (
Following OVA sensitisation and short or long term OVA challenges or after sham sensitisation and long or short term PBS challenges, concentrations of (a) IL-5, (b) IL-13 and (c) activated TGF-β1were measured in BAL fluid by ELISA 3 days to 8 weeks after final airway challenge, n≥15 per group from 5 independent experiments. Significant differences: * OVA challenges versus PBS controls, +long term versus short term OVA challenges, */+ p<0.05, **/++ p<0.01, ***/+++ p<0.001.
In addition, we assessed intracellular cytokines in lung mononuclear cells. Four weeks after long term but not short term challenges, percentages of IFN-γ producing lung cells (5.31±0.45%) were significantly elevated compared to long term PBS controls (3.64±0.55%, p<0.01, n = 10, 2 independent experiments). Similar differences 3 days and 2 weeks after long term challenges did not reach significance. Further, the percentage of IL-5 producing lung cells were significantly elevated following OVA challenges compared to PBS challenged controls and IL-5 producing lung cells were significantly more common three days and two weeks after long term OVA challenges (5.43±0.72% and 5.78±0.36%, p<0.01, n = 10) than after short term exposure (3.76±0.44% and 3.28±0.29%, both n = 10). IL-4+ lung cells were detectable after OVA challenges but there were no differences between protocols (data not shown).
In addition to the cytokines mentioned above we also assessed BAL levels of activated TGF-β1, a cytokine involved in the development of fibrosis. TGF-β1 was not detectable in control mice, but was markedly elevated 3 days after both short and long term challenges (
Systemic OVA sensitisation with consecutive OVA challenges induced OVA-specific IgE in serum without significant differences between long and short term challenges (
Short term OVA challenges | Long term OVA challenges | |||||
Time post challenge | IgE [IU/ml] | IgG1 [IU/ml] | IgG2a [IU/ml] | IgE [IU/ml] | IgG1 [IU/ml] | IgG2a [IU/ml] |
3 days | 34.6±11.7** | 0.00±0.00 | 210±78* | 373.9±96.7**/++ | 72.18±37.14*/+ | |
2 weeks | 37±10** | 310.6±73.9** | 0.01±0.01 | 60±16** | 433.5±91.8** | 18.39±9.20*/+ |
4 weeks | 56±13*** | 138.4±64.5 | 4.66±4.40 | 96±39* | 254.5±51.2*** | 21.6±10.11*/+ |
8 weeks | 112±58* | 92.2±57.3 | 40.03±11.52* | 102±47* | 238.4±63.9*** | 130.10±39.85**/+ |
Serum concentrations of OVA-specific antibodies post challenges were assessed by ELISA. In PBS controls no OVA-specific IgG1 or IgG2a was detected and there was a background of OVA-specific IgE of maximally 1.0 IU/ml. Significant differences: *versus respective PBS control, +versus respective time point after short term OVA challenges, */+ p<0.05, **/++ p<0.01, ***/+++ p<0,001. Means±SEM are shown, n≥9 from 3 independent experiments.
The pathogenic mechanisms involved in the development of chronicity in asthma are poorly understood. Recently established murine models of allergen sensitisation followed by long term allergen challenges do not only lead to impaired lung function (similar to short term challenge models) but also to airway wall remodelling, a feature typical of chronic asthma. Only a few of these studies investigated the duration of changes after the end of challenges and yielded conflicting results
Lung function impairment with airflow limitation is thought to be the most threatening consequence of severe asthma. It is therefore of major importance to identify mechanisms involved in the development of chronically impaired lung function. In the present model, AHR was only short lived after cessation of challenges as observed previously
Increases in airway collagen deposition, are thought to contribute to the development of chronic lung function impairment. This notion was supported by a report from a chronic asthma model that depletion of T-cells did not abrogate AHR
Goblet cell hyperplasia, another feature of airway wall remodelling, developed during challenges and thus might play a role in the induction of lung function changes. However, AHR resolved when goblet cell hyperplasia was still marked suggesting that mucus overproduction does not necessarily result in AHR. Interestingly, goblet cell hyperplasia and baseline lung function changes resolved around the same time between 4 and 8 weeks after long term challenges, suggesting a possible link.
Airway allergen challenges induced long lasting airway inflammation in our model with striking differences depending on the duration of challenges. Sustained airway lymphocytosis and moderate, short lived eosinophilia were induced by long term challenges, while short term challenges resulted in marked eosinophilia which resolved after 2 weeks. The extent of airway eosinophilia has been related to the degree of AHR in asthmatics
The Th2 cytokines IL-5 and IL-13 have been implicated in the development of AHR in many models of short term allergen challenges. Following long term challenges we found persistent IL-5 production in the airways and low, transient levels of IL-13. In contrast, short term challenges were associated with strong but transient IL-13 production and low levels of IL-5. Predominant IL-13 production after short term, but not after prolonged challenges has been seen in similar models of OVA sensitisation
In addition to Th2 responses, Th1 cytokines have been detected in severe asthma
TGF-β1, a cytokine known to induce fibrosis, is produced by a range of airway and immune cells including a subset of regulatory T cells
In summary, we report a mouse model of sensitisation followed by long term allergen challenges leading to sustained allergic airway inflammation, goblet cell hyperplasia, peribronchial fibrosis, and impaired lung function. The sequence in which these features arise and resolve suggests that airway wall fibrosis may not contribute to lung function impairment. The marked differences in the quality of airway inflammation and immune responses after long and short term allergen challenges may help to elucidate mechanisms responsible for chronic functional and structural changes in asthma.
We thank Dr Michael Hollinshead, Imperial College London, for his advice regarding the analysis of peribronchial collagen deposition.